The Binding Site in β2-Glycoprotein I for ApoER2′ on Platelets Is Located in Domain V

Abstract

The antiphospholipid syndrome is caused by autoantibodies directed against ␤ 2 -glycoprotein I (␤ 2 GPI). Dimerization of ␤ 2 GPI results in an increased platelet deposition to collagen. We found that apolipoprotein E receptor 2 (apoER2 ), a member of the low density lipoprotein receptor family, is involved in activation of platelets by dimeric ␤ 2 GPI. To identify which domain of dimeric ␤ 2 GPI interacts with apoER2 , we have constructed domain deletion mutants of dimeric ␤ 2 GPI, lacking domain I (⌬I), II (⌬II), or V (⌬V), and a mutant with a W316S substitution in the phospholipid (PL)-insertion loop of domain V. ⌬I and ⌬II prolonged the clotting time, as did full-length dimeric ␤ 2 GPI; ⌬V had no effect on the clotting time. Second, ⌬I and ⌬II bound to anionic PL, comparable with full-length dimeric ␤ 2 GPI. ⌬V and the W316S mutant bound with decreased affinity to anionic PL. Platelet adhesion to collagen increased significantly when full-length dimeric ␤ 2 GPI, ⌬I, or ⌬II (mean increase 150%) were added to whole blood. No increase was found with plasma ␤ 2 GPI, ⌬V, or the W316S mutant. Immunoprecipitation indicated that full-length dimeric ␤ 2 GPI, ⌬I, ⌬II, and the W316S mutant can interact with apoER2 on platelets. ⌬V did not associate with apoER2 . We conclude that domain V is involved in both binding ␤ 2 GPI to anionic PL and in interaction with apoER2 and subsequent activation of platelets. The binding site in ␤ 2 GPI for interaction with apoER2 does not overlap with the hydrophobic insertion loop in domain V.The antiphospholipid syndrome is a non-inflammatory autoimmune disease associated with a wide variety of clinical symptoms. The main clinical features are arterial, venous, or small vessel thrombosis, both early and late pregnancy losses, and pre-eclampsia (1-4). The syndrome is diagnosed when one of the above clinical criteria is accompanied by the persistent presence of antiphospholipid antibodies (aPL) 3 (lupus anticoagulants and anticardiolipin antibodies) in the plasma of patients. These aPL are a heterogeneous group of antibodies directed to plasma proteins with affinity for anionic phospholipids (PL). We now know that the most important plasma protein, to which the aPL are directed, is ␤ 2 -glycoprotein I (␤ 2 GPI or apolipoprotein H) (5, 6).␤ 2 -Glycoprotein I is abundantly present in plasma (ϳ200 g/ml) and is mainly synthesized in the liver, although mRNA coding for ␤ 2 GPI has been found in a variety of cells such as trophoblasts, placental cells, endothelial cells, and neurons (7-9). The mature sequence of human ␤ 2 GPI consists of 326 (44 kDa) amino acids (aa) with four N-linked glycosylation sites. It is composed of five repeating units that belong to the complement control protein family. The first four domains have ϳ60 aa residues and 4 cysteines each, with potential disulfide bridges joining the first to third and the second to fourth cysteines to contribute to a "looped-back " structure, called Sushi domains. The fifth domain is aberrant, having 82 aa and three disulfide bridges. A...

Antiphospholipid antibodies promote leukocyte–endothelial cell adhesion and thrombosis in mice by antagonizing eNOS via β2GPI and apoER2

“…In selected experiments, the cells were treated with anti-β2GPI monoclonal antibody (FC1, 3F8, or 2aG4; 10 μg/ml). Additional experiments were performed in cells pretreated with recombinant monomeric β2GPI (apple 2-β2GPI, 1.5 μg/ml) or dimerized β2GPI (apple 4-β2GPI, 1.5 μg/ml), which were was constructed and expressed as described previously (15,38). Studies were also done to assess the role of LDLR family members in cells pretreated with GST-RAP or GST control (12 μg/ml for 15 minutes), which were purified as previously reported, and purity was confirmed by SDS-PAGE (36).…”

“…In addition, we determined whether aPL-induced eNOS inhibition involves β2GPI, and if the process also requires an LDL receptor (LDLR) family member, particularly apoER2, which has the capacity to directly bind β2GPI (14,15). Complementary experiments evaluating eNOS activation and leukocyte-endothelial cell adhesion were performed to link changes in enzyme activity with alterations in a key endothelial cell function that contributes to both the proinflammatory and the prothrombotic actions of aPL (16).…”

The significance of autoantibodies against β2-glycoprotein I

“…Biochemical analysis has shown that domain V interacts with the first LDL type A (LA) module of LRP8. 109 Solution nuclear magnetic resonance spectroscopy has identified the residues in both LRP8 and domain V of ␤ 2 GPI that are perturbed when both proteins interact. 110 Interestingly, the epitope in domain V that interacts with LA1 of LRP8 involves several positively charged residues, including the positively charged patch of lysine residues that are essential for phospholipid binding.…”

Current concepts on the pathogenesis of the antiphospholipid syndrome

“…This would be somewhat analogous to the antichromatin Ab/chromatin immune complex driving additional autoantibody production in an SLE murine model via a crosslinking mechanism involving TLR9 and the B-cell receptor. 99 Direct binding experiments, as have been performed to study the interaction between ␤ 2 -GPI and other receptors such ApoER2Ј, 100 annexin A2, 64 and GPIb␣, 27 are required to solidify the involvement of TLR4.…”